Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves

Abstract

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 ± 10, mean ± SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (ε = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 μmol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin π cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.